Fluorescent dyes or stains can be used in the detection of nucleic acids, such as DNA, RNA, DNA/RNA hybrid molecules, and biological samples containing the same. Nucleic acid polymers, such as DNA and RNA hold the genetic information that is transmitted from one generation to the next. These molecules are also responsible for maintaining routine function of a living organism. Nucleic acids are thus of interest and the objects of study. Fluorescent nucleic acid dyes that specifically bind to nucleic acids and form highly fluorescent complexes are useful tools for such study. These dyes can be used to detect the presence and quantities of DNA and RNA in a variety of media, including pure solutions, cell extracts, electrophoretic gels, micro-array chips, live or fixed cells, dead cells, and environmental samples.
Nucleic acid staining is generally performed using one of the three major methods: 1) prestaining; 2) in-gel or precast staining; and 3) post staining. In the pre-staining method, a nucleic acid binding dye is pre-mixed with a nucleic acid sample to form DNA- or RNA-dye complexes in a loading buffer. The resulting solution is then loaded into a well in a gel for gel electrophoresis. During electrophoresis, the DNA- or RNA-dye complexes migrate through the gel matrix and separate into bands according to their molecular sizes. In the in-gel staining method, the dye is embedded throughout the gel matrix by adding the dye to the gel-forming material (e.g., agarose powder and buffer) when the gel is poured. During gel electrophoresis, the migrating nucleic acids encounter the dye in the gel matrix to form fluorescent nucleic acid-dye complexes. In post staining method, a nucleic acid sample is first separated by gel electrophoresis. The gel containing the separated bands is then immersed in a solution containing the nucleic acid dye to allow the formation of nucleic acid-dye complex. Depending on the dye used, a washing or de-staining step may be necessary for some of the dyes in order to remove the background.
There are advantages and disadvantages associated with each of the methods. The prestaining method is generally more desirable because it requires less dye molecules and thus minimizes the chance for a handler to exposure to potentially toxic dye molecules. It also offers flexibility by permitting any unused wells in the gel to be used again later. Furthermore, it saves time by eliminating an extra staining step. However, most nucleic acid dyes when used in conventional buffer systems are not suitable for this method, either because the dyes do not have sufficient binding affinity to accompany the nucleic acid molecules during migration, or because the binding is too tight which causes retardation of nucleic acid migration. Most nucleic acid dyes are positively charged and thus they tend to migrate in the opposite direction, which makes detection of the faster-moving small nucleic acid molecules insensitive. Even for dyes that have been used for the prestaining method, the aforementioned problems still exist to a certain degree, not to mention safety issues. In theory, in-gel staining is the next best choice because no extra staining step is required. However, poor band resolution caused by dye interference to nucleic acid dye migration often limits its utility. Like prestaining, dye migrating to the opposite direction can also be a problem. Post gel staining requires an extra staining step, which is a disadvantage, especially if the dye is toxic. However, post staining also has a major advantage in that nucleic acid migration is never interfered by the dye, thus it often can result in good gel resolution even when conventional buffers are used.
A variety of nucleic acid stains have been used for detecting nucleic acids in gels. The classic and still the most widely used nucleic acid gel stain is ethidium bromide (EB). The dye is inexpensive and offers sufficient sensitivity for most applications. A major problem associated with EB, however, is its toxicity. EB is known to be a powerful mutagen. As a result, special handling and disposal are required for the dye, making the dye ultimately more expensive to use.
In recognition of the problem, alternative dyes to EB have been developed in recent years. These dyes include SYBR Green I and SYBR Safe (US patent Application No. 2005/0239096) from Invitrogen, Co. and GelRed and GelGreen (U.S. Pat. No. 7,601,498) from Biotium, Inc. These gel stains have been shown to be either weakly mutagenic or completely nonmutagenic by standard Ames test. Moreover, SYBR Safe, GelRed and GelGreen have also been tested to be safe to aquatic life, making the dyes easy to dispose. However, although SYBR Green I and SYBR Safe have improved mutagenic safety profile, they are still cytotoxic at relatively high concentrations due to their relatively small molecular sizes, which facilitate their rapid entry into cells (Briggs C and Jones M, Acta Histochem. 107(4), 301(2005)).
GelRed and GelGreen belong to a new class of dimeric nucleic acid dyes with a flexible neutral linker (U.S. Pat. No. 7,601,498). On average, these dimeric dyes have a molecular weight at least 2-3 times that of SYBR Safe or SYBR Green I and bear two positive charges as opposed to only one positive charge for SYBR Safe, for example. The much larger sizes as well as the higher charge of GelRed and GelGreen render them difficult to cross the cell membranes, thus denying the opportunity for the dyes to interfere with any intracellular activities, including activities associated with genomic DNA. Consequently, GelRed and GelGreen are not only nonmutagenic but also noncytotoxic within the dye concentration range normally used for nucleic acid gel staining. Furthermore, dimeric dyes such as GelRed and GelGreen exhibit exceptional signal-to-noise ratio because the dyes self-quench in the absence of nucleic acids to result in very low background fluorescence. The improved safety and sensitivity of GelRed and GelGreen make them some of best nucleic acid gel stains.